The TSH receptor (TSH-R) plays a key role in function and growth of thyroid cells. This receptor is a member of a subfamily of G-protein-coupled glycoprotein receptors, which additionally in particular also comprise receptors for the luteinizing hormone/chorionic gonadotropin (LH/CGR) and the follicle-stimulating hormone (FSHR). The receptors of this subfamily have a large N-terminal extracellular domain, which is of essential significance for ligand binding, and for which it was shown, that it is involved in signal transfer. The transfer of the TSHR signal is mainly mediated by activating adenylate cyclase, which results in an increase of the intracellular cAMP level.
Part of the large interest in the TSH receptor is to be attributed to its role as primary auto-antigen for thyroid gland autoimmune diseases, which are accompanied by the occurrence of auto-antibodies against the TSH receptor. Such thyroid gland autoimmune diseases in particular include Basedow's disease, an autoimmune disease resulting in hyperthyroidism, which is one of the most frequent human autoimmune diseases. Basedow's disease is caused by activation of adenylate cyclase and the resulting cAMP increase. This results in hyperthyroidism, goiter formation, and possibly eye changes. The auto-antibodies against the TSH receptor can also be of a blocking nature, and thus inhibit adenylate cyclase and cAMP. In this case, there is a hypofunction of the thyroid gland. Simultaneous occurrence of stimulating and blocking auto-antibodies in the affected patients is likewise possible, wherein the portion of stimulating antibodies usually predominates.
For detection of such autoimmune antibodies, there has been a bioassay for some time now, in which the cAMP increase is measured. This measuring method is very time-consuming. Furthermore, the bioassay is not reliable, since it can provide false-positive results. Within the scope of this description, this type of measuring method will be designated as bioassay to differentiate it from the in-vitro methods for determination of autoimmune antibodies against the TSH receptor. In an in-vitro detection method for autoimmune antibodies available on the market, a TSH receptor extracted from pork thyroid gland membrane is used (first generation of in-vitro methods). In another assay for detection of autoimmune antibodies against the TSH receptor, a complete human recombinant TSH receptor protein (wild type) is used in a competition assay (second generation of in-vitro methods).
Thyroid, Vol. 7 (1997) 867-877 describes the epitopes for stimulating and blocking antibodies at the TSH receptor. The majority of the functional epitopes for stimulating antibodies are located in the range of amino acids 8 to 168, and those for the blocking antibodies in the range of amino acids 261 to 370 of the receptor protein. For activity measurements, the above stated bioassay is used.
WO 01/27634 A1 for the first time provides a quantitative method for simultaneous detection of autoimmune antibodies of different specificity, which is quickly reproducible and executable with high accuracy. For this purpose, TSH receptor chimeras are used, which differ from the wild type receptor in that individual sequences, to which autoimmune antibodies bind, are substituted with respective sequences of another receptor from the class of G-protein-coupled receptors. The TSH receptor chimeras are based on the complete TSH receptor protein. However, the measuring effort is high. The separation by centrifugation makes the method too cumbersome for routine use. The assay also must be executed in an ice bath or at 4° C., since the TSH receptor chimeras are not very stable. A respective technical teaching can be found in WO 01/63296 A1, where the use of a sandwich technique is suggested for detection. It turned out, however, that usually with the assay materials suggested there, unspecific binding is too high. An assay for detection of autoimmune antibodies on the basis of the findings of the two patent applications stated above is not available on the market.
For the purpose of this description, a measuring method for detection of autoimmune antibodies available on the market for some time now, which uses the complete unchanged TSH receptor in a competition assay, will be designated as second-generation detection method. This assay is suitable for detection of Basedow's disease. It is, however, disadvantageous that stimulating, blocking and neutral autoimmune antibodies cannot be distinguished. Furthermore, not all subtypes are identified, since the displaced TSH only binds to the epitope for stimulating and blocking auto-antibodies at 30 to 40%.